2. The Baltimore Scheme Flashcards
what information is encoded in a viral genome? (5)
gene products to complete its life cycle:
1. replication of viral genome
2. assembly and packaging of genome
3. regulation and timing of replication cycle
4. modulation of host defenses
5. spread to other cells
what is the main principle of the baltimore scheme?
viral genomes MUST make mRNA to be read by host ribosomes
what does positive polarity of a DNA or RNA strand mean?
coding strand
what does negative polarity of a DNA or RNA strand mean?
non-coding/template/complementary
describe the polarity of mRNA
POSITIVE –> bc ready to be read by ribosomes
are all positive RNAs mRNA?
NO –> not all are translated
ex. genomic RNA
why is + RNA in retroviruses not mRNA?
retroviruses are + RNA viruses that use RT to make DNA that integrates into the host to make mRNA
What are the 7 types of viruses in the baltimore scheme?
- dsDNA
- ssDNA
- dsRNA
- (+) ssRNA
- (-) ssRNA
- (+) ssRNA-RT
- dsDNA-RT
- describe dsDNA viruses
dsDNA is copied by DNA polymerases and mRNA is made directly
what are the 2 types of dsDNA viruses?
- genomes copied by host DNA polymerase
- genomes that encode their own DNA polymerase
- describe ssDNA genomes
genome is circular or linear
(+) or (-) converted to dsDNA with DNA polymerase to produce mRNA with host transcription machinery
why do RNA viruses replicate in the cytoplasm?
they cannot use the host RNA polymerase to make mRNA bc it uses DNA as a template –> requires its own polymerase to make mRNA from RNA
which types of viruses use RdRP? how does RdRP work?
dsRNA, (+) RNA, and (-) RNA viruses encode RdRP to make RNA and mRNA from RNA
which types of viruses use RT? how does RT work?
ssRNA-RT and dsDNA-RT viruses encode RT to make DNA tha is transcribed by the host RNA pol II to make mRNA
- describe dsRNA genomes
dsRNA must be copied to mRNA using RdRP* bc ribosomes can’t translate dsRNA
*viruses carry RdRP in the viral particle
- describe (+) ssRNA genomes
directly translated –> doesn’t need to carry RdRP in the particle bc RNA can be translated in the host to make RdRP and make MORE (+) ssRNA
- describe (-) ssRNA genomes
cannot be translated so must carry RdRP to make mRNA
- describe (+) ssRNA-RT genomes
it is positive but cannot be translated so must be copied by RT* to (-) ssDNA then to dsDNA –> dsDNA integrates into the host DNA (PROVIRUS) by integrase to be transcribed by host RNA pol to make mRNA
*RT is carried in the viral particle
- describe dsDNA-RT genomes
the dsDNA is gapped (cannot be copied to mRNA) –> converted to full dsDNA then mRNA
the +RNA is then converted to -DNA to remake gapped dsDNA by RT
what happens when multiple viruses co-infect the same cell?
viral genome segments undergo REASSORTMENT –> genomes get mixed up during replication to cause rapid gain in evolution
why are lab animals good?
to study pathogenesis
how were viruses propagated before cell culture? how does this work?
in embryonated chicken eggs
- 5-14 days after fertilization a hole is drilled in the shell and virus is injected –> to make vaccine
what was the first virus they found could multiply in cultured cells?
poliovirus
what are the 2 types of cell culture?
- primary cell culture –> prepared from animal tissues, limited life span
- continuous cell culture –> single cell type that propagates indefinitely
what is the benefit of using primary cell cultures?
more biologically accurate –> these are the actual cell types being infected by the virus
example of continuous cell lines
HeLa cells –> tumour tissue
what is the downside of using continuous cell lines?
may not resemble cell of origin
what are cytopathic effects? and some examples?
CPE are evidence of viral growth in cultured cells
ex. detachment, lysis, syncytium formation (cell fusion), nuclear shrinking/swelling, accumulation of virions/viral proteins, altered membrane
describe the PLAQUE ASSAY
- what does it assume?
to quantify the number of infectious particles –> as PFU
assumes 1 viral particle produces a plaque –> 1 cell is infected and lysed, then neighbouring cells get infected to form a plaque
what is virus titer?
concentration of a virus in a sample
how do you calculate the virus titer?
find titer of 10^(-6) dilution –> #PFU/vol
ex. 17 PFU/0.1ml = 170 PFU/ml
then, find titer of undiluted stock –> dilution titerdilution
ex. 170 PFU/ml10^6 = 1.7x10^8 PFU/ml
describe the FOCUS-FORMING UNIT ASSAY
to find # of infectious particles for viruses that don’t lyse cells (i.e. don’t create plaques)
after infection, cells are permeabilized and stained with antibody against viral protein
what is the particle-to-PFU ratio? why do we use it?
of actual viral particles / # of infectious particles(PFU)
we use this because not all particles can cause infection
what would cause a viral particle to not be able to initiate an infection?
damaged, mutations, “empty”, complexity of life cycle, anti-viral defense, missing part of genome
what is the multiplicity of infection (MOI)?
average number of infectious particles ADDED per cell
(**not the same as # of infectious particles a cell receives)
how do you calculate MOI?
EX. add 10^7 PFUs to 10^6 cells –> 10 particles/cell ADDED –> MOI = 10
What does MOI = 10 indicate?
each cell does NOT receive 10 virions –> infection is due to random collisions so some cells be uninfected and some can receive diff numbers of particles
describe the distribution of virus particles per cell
poisson distribution
what happens if you use a low MOI
viruses < cells
- allows you to have multiple cycles of infection
what happens if you use an optimal MOI
viruses ~ cells
- have most cells infected
what happens if you use a high MOI?
viruses > cells
- when every cell needs to be infected
